It has been well established that all positive-strand RNA viruses of eukaryotic cells, including poliovirus and hepatitis C virus (HCV), replicate their genomes on the cytosolic surface of membranous structures. However, the precise mechanisms of specific priming, strand separation, and RNA elongation during viral RNA synthesis remain to be elucidated. Current reconstituted systems used to characterize poliovirus and HCV RNA replication fail to mimic specific transcription initiation and elongation of viral RNA, suggesting the absence of an essential structural or functional component. In fact, most of these systems lack an important constituent: lipid bilayers. The tethering of viral RNA replication complexes to the cytosolic surface of intracellular membranes could affect several aspects of their structure and specific activity. Therefore, the proposed research will determine the effect of membrane association on the architecture of protein-protein interactions and on the specific activity of the poliovirus and HCV RNA-dependent RNA polymerases by: Specific Aim 1: To develop a new model membrane system to study poliovirus RNA replication;Specific Aim 2: To identify novel viral and cellular components of the poliovirus RNA replication complex;Specific Aim 3: To develope a model membrane system to study hepatitis C virus RNA replication. Poliovirus or HCV RNA replication proteins will be reconstituted in the presence and absence of synthetic and natural membranous platforms. The effect of membrane support on polymerase function will be tested by determining the specific activity and kinetics of the viral polymerase. The structure of the reconstituted proteins will be visualized by electron microscopy and assessed using mutant viral polymerases with specific defects in potential molecular interfaces. PUBLIC HEALTH RELEVANCE: Positive-strand RNA viruses are responsible for a wide range of disease worldwide. Although a vaccine exists to prevent poliovirus infection and the development of poliomyelitis, a vaccine has not yet been developed for numerous other positive-strand RNA viruses, including HCV, the leading cause of hepatocellular carcinoma. Understanding the replicative cycle of HCV and poliovirus will facilitate the identification and effective regulation of antiviral targets to prevent diseases caused by these viruses.